The long-range goal of these studies is to understand the precise function of osteocalcin, the vitamin-K dependently synthesized protein of bone. Osteocalcin is the major noncollagenous protein in bone matrix and the fifth most abundant skeletal protein. The protein is synthesized de novo in bone tissues and appears to be localized to those cells expressing osteoblastic phenotype. Since its discovery in 1976, much knowledge has accumulated regarding the physical and molecular properties of osteocalcin and the potential use of this bone specific protein as a sensitive marker of bone turnover by measurement of its concentration in the circulation. Our studies in the preceding three years have brought to realization two important facts that could lead us to define the function of oseteocalcin: 1) Gamma-carboxyglutamic acid concentration in bone precisely correlates to calcium concentration, 2) high molecular weight Gla-protein(s) occur in bone with weak immunoreactivity to osteocalcin, and 3) the biosynthesis of osteocalcin is "modulated" by the 1,25(OH)2D3 metabolite. The putative precursors of osteocalcin (high molecular weight Gla protein) will be characterized in warfarin-treated embryonic bone cultures, microsomal preparations and vitamine D deficient since all three systems have been demonstrated to accumulate Gla-protein or substrate and be immunologically reduced in 6000K molecular weight osteocalcin peptide. The dependency of vitamin D for either de novo synthesis of osteocalcin and/or molecular processing of pro-osteocalcin will be examined in vitro in bone organ culture and in microsomal preparations. Further the relationship of osteocalcin response to 1,25(OH)2D3 as reflecting osteoblast proliferation or the mobilization of calcium from bone will be explored in vitro.